Mass spectrometry (MS) is arguably among the most useful detection schemes for capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) largely due to the limited information obtained with other common detection techniques, such as UV, visible, and fluorescence spectrometry. The limits of these common detection techniques are particularly evident in the identification and analysis of macromolecules, such as peptides and proteins. While CE is itself used as an analytical method, CE has also been utilized to separate analytes prior to analysis by both fast atom bombardment (Moseley, M. A., Deterding, L. J., Tomer, K. B., Jorgenson, J. W., J. Chromatog., 1989, 480, 197) and matrix assisted laser desorption ionization MS instrumentation (Preisler, J., Foret, F., Karger, B. L., Anal. Chem., 1988, 70, 5278). In addition, a very useful technique is obtained by interfacing CE directly online with electrospray ionization (ESI) mass spectrometry, referred to as “CE-ESI-MS”.
The successful operation of a CE-ESI-MS system typically requires a closed circuit for both the CE separation and the electrospray ionization processes. Three major designs have been advanced, namely, coaxial sheath flow (Smith, R. D., Barinaga, C. J., Udseth, H. R., Anal. Chem., 1988, 60, 1948), liquid junction (Lee, E. D., Muck, W., Henion, J. D., Covey, T. R., Biomed. Environ. Mass. Spectrom., 1989, 18, 844), and sheathless flow (Olivares, J. A., Nguyen, N. T., Yonker, C. R., Smith, R. D., Anal. Chem., 1987, 59, 1230). Coaxial sheath flow is the basis of most commercial instruments, though it suffers in sensitivity. The low sensitivity of a coaxial sheath flow CE design arises largely from the relatively high sheath flow compared to the flow from the CE capillary, resulting in not only a large dilution of the eluting analytes, but also in hindered desorption of ions due to the non-optimal electrospray that results at such high flow rates. Coupling CE online with MS through a liquid junction arrangement requires tedious capillary alignment and end-to-end butting of the separation capillary and the spray tip. Unfortunately, even under the best conditions, sensitivity is compromised by loss and spreading of sample analytes in the relatively large dead volume of the liquid junction.
Electrolytic interfaces for coupling electrophoresis capillaries to electrospray tips (spray tips) have been designed for conveying analyte ions to mass spectrometers. Such interfaces have been designed to effect completion of an electrolytically conductive fluid circuit in the capillary tube, which include openings near the spray tip, and physical breaks connected by permeable sheaths near the spray tip. Unfortunately, these breaks and openings result in analyte loss, disruption of the fluid flow path, disruption of the electric field, or a combination of these effects near the spray tip, which ultimately degrades mass detection sensitivity.
Sheathless flow is, in principle, a desirable design for coupling CE online with MS, one reason being that analyte dilution is minimized compared to capillaries incorporating a sheathed design. A variety of sheathless designs have been described that satisfy the requirement of closing the CE separation capillary circuit while simultaneously providing an electrical potential to the spray tip. These, for example, include the use of a single capillary whereby electrical contact is established through: (a) coating the capillary outlet with a conductive metal (Olivares, J. A., Nguyen, N. T., Yonker, C. R., Smith, R. D., Anal. Chem., 1987, 59, 1230; Kelly, J. F, Ramaley, L, Thibault, P., Anal. Chem., 1997, 69, 51; Barraso, M. B., deJong, A. P., J. Am. Soc. Mass Spectrom., 1999, 10, 1271; Chang, Y. Z., Her, G. R., Anal. Chem., 2000, 72, 626; Figeys, D., Oostveen, I., Ducert, A., Aebersold, R., Anal. Chem., 1996, 68, 1822; Kriger, M. S., Cook, K. D., Ramsey, R. S., Anal. Chem., 1995, 67, 385; Wilm, M., Mann, M., Anal. Chem., 1996, 68, 1) or polymer (Maziarz, E. P., Lorentz, S. A., White, T. P., Wood, T. D., J. Am. Soc. Mass Spectrom., 2000, 11, 659); (b) insertion of a conductive wire into the outlet of the capillary (Fang, L., Zhang, R., Williams, E. R., Zare, R. N., Anal. Chem., 1994, 66, 3696) or through a small pinhole in the wall of the capillary (Cao, P., Moini, M., J. Am. Soc. Mass Spectron., 1998, 9, 1081; Smith, A. D., Moini, M., Anal. Chem., 2001, 73, 240); (c) splitting the capillary effluent at or near the capillary outlet to fill the gap between the capillary and an outer coaxial metallic sleeve (Moini, M. Anal. Chem. 2001, 73, 3497, Petersson, M. A., Hulthe, G., Fogelqvist, E., J. Chromatogr. A, 1999, 854, 141), or (d) adjusting the position of the outlet of the capillary such that electrical contact is established through the air to the grounded inlet capillary of the MS (Mazereeuw, M., Hofte, A. J. P., Tjaden, U. R., van der Greef, J., Rapid Commun. Mass Spectrum., 1997, 11, 981). Another strategy for the fabrication of a sheathless interface is to use two pieces of capillary whereby the CE capillary is connected to a short spray tip via a sleeve. The sleeve could be a piece of microdialysis tubing (Severs, J. C., Smith, R. D., Anal Chem., 1997, 69, 2154), stainless steel tubing (Figeys, D., Ducret, A., Yates, J. R., Aebersold, R., Nature Biotechnol., 1996, 14, 1579), or a micro-tee (Tong, W., Link, A., Eng, J. K., Yates, J. R., Anal. Chem., 1999, 71, 2270). Although these approaches do produce operational interfaces, their fabrication requires delicate manipulation of miniaturized components and they suffer in their robustness. In the two piece approach, the CE separated zones are invariably broadened at the junction between the separation column and the tip, since the inside diameter of the sleeve has to be larger than the outside diameter of the separation capillary. Furthermore, these junctions often suffer from misalignment and imperfect butting of the two pieces of capillary. Single capillary methods appear to disrupt the CE separation the least, however, metal coatings on fused silica capillaries are not durable and drilling pin-holes through capillary walls is a delicate and irreproducible procedure. Once operational, a split-flow interface is highly sensitive, as demonstrated by Moini et al. who reported the separation and detection of proteins from human red blood cells at attomole levels (Moini, M., Demars, S. M., Huang, H., Anal. Chem., 2002, 74, 3772). Sheathless interfacing has been the subject of several recent reviews, where the advantages and limitations of this design have been enumerated (Tong, W., Yates, J. R., Chromatographia Supplement, 2001, 53, S90; Ding, J., Vouros, P., Anal. Chem., 1999, 71, 378A; Gelpi, E., J. Mass Spectrum., 2002, 37, 241; Moini, M., Anal. and Bioanal. Chem., 2002, 373, 466).
Thus, there is a need to provide improved capillary designs for coupling CE with MS that overcome these problems.